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Biomedical subjects

G Camussi

Publications and source records attributed to G Camussi.

At least 73 records · Page 4Linked to original sources
PubMed1997

Persistent infection of human vascular endothelial cells by group B coxsackieviruses.

Group B coxsackieviruses (CVBs) cause >20% of the cases of myocarditis and dilated cardiomyopathy. Information on the permissiveness of vascular cells to CVBs is scant. Interactions of CVBs with human vascular endothelial cells (ECs) were investigated in vitro. All 6 CVBs (CVB-1 to -6) consistently infected primary EC cultures and an immortalized EC line without producing cytopathology. Whereas replication of types 1, 2, 4, and 6 ceased within 30-60 days after infection, CVB-3 and -5 caused a persistent infection. Replication of CVB-3 and -5 continued for >260 days. In ECs, the constitutive production of interferon-beta, but not of other cytokines, appeared to confer resistance to CVBs. Persistence of CVB-3 and -5 was associated with the chronic release of tumor necrosis factor-alpha, a cytotoxic cytokine that also has a negative inotropic effect on myocardial cells. The results suggest that chronic endothelial CVB infections may play a role in vascular disease.

Cells, Cultured↗
PubMed1997

Interleukin-1 receptors and receptor antagonist in haemodialysis.

BACKGROUND: Biological activity of interleukin-1 (IL-1) depends on the number and type of IL-1 receptors on target cells and on the amounts of its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1ra). METHODS: Expression of IL-1 receptor was studied on the peripheral blood mononuclear cells of 20 end-stage renal-disease patients maintained by chronic haemodialysis by means of either polysulphone (10 patients) or cuprophane membranes (10 patients) and compared to that of normal controls. Plasma and cellular levels of IL-1ra and IL-1 beta were also measured. RESULTS: The proportion of monocytes expressing the IL-1 receptor was strikingly higher in haemodialysis patients than in the healthy population. This proportion further increased during haemodialysis with cuprophane but not with polysulphone. Expression of the IL-1 receptor on lymphocytes was very low in both controls and dialysed patients; in the latter there was no intradialytic variation. Plasma concentrations of IL-1 beta and IL-1ra were elevated in haemodialysis patients and undetectable in controls. Whereas plasma IL-1 beta decreased throughout haemodialysis, IL-1ra further increased, with no significant differences between the two membranes used. Total cellular IL-1 beta and IL-1ra were also higher in the patient group than in the healthy controls. A further increase of both IL-1 beta and IL-1ra was detected at the end of the haemodialysis session with any membrane. CONCLUSIONS: Monocytes of haemodialysis patients circulate in a state of activation, which makes them both producer and target of IL-1. Thus there is an autocrine upregulation of IL-1 production. Although IL-1ra levels are high, they are most likely to be expression of monocyte activation rather than represent effective inhibitors of IL-1 activity.

Adult↗
PubMed1997

Alternative pathway complement activation induces proinflammatory activity in human proximal tubular epithelial cells.

BACKGROUND: Proximal tubular epithelial cells express a surface C3-convertase activity which induces C fixation and insertion of the C5b-9 membrane attack complex (MAC) into the cell plasma membrane. The physiopathological consequences of this phenomenon are unknown. METHODS: The effect of C fixation on the production of inflammatory mediators by human proximal tubular epithelial cells in culture was explored. RESULTS: Proximal tubular epithelial cells incubated with a sublytic amount of normal human serum as a source of C, but not with heat-inactivated human serum, showed a time-dependent calcium influx and a concomitant release of 14C-arachidonic acid (14C-AA). Eicosanoid synthesis following the arachidonic acid mobilization was studied as prostaglandin E2 release. Mg2+/EGTA, which did not prevent C activation by the C3-convertase, and p-bromodiphenacyl bromide a phospholipase A2-inhibitor, inhibited mobilization of 14C-AA. These results suggest the activation of an extracellular Ca(2+)-dependent, phospholipase A2. Complement fixation was associated with the synthesis of proinflammatory cytokines such as IL-6 and TNF-alpha. Experiments with C6-deficient sera indicated that the release of 14C-AA and the production of cytokines were dependent on the insertion of the terminal components of complement in the plasma membrane. Indeed, the reconstitution of normal haemolytic activity of C6-deficient sera with purified C6 restored also the release of 14C-AA and the production of cytokines. CONCLUSIONS: In vitro complement activation on the proximal tubular cell surface triggers the generation of proinflammatory mediators, which may potentially contribute to the pathogenesis of tubulointerstitial injury.

Arachidonic Acid↗
PubMed1997

Modulatory effect of interleukin-10 on the production of platelet-activating factor and superoxide anions by human leucocytes.

We observed that human monocytes (MO) and polymorphonuclear neutrophils (PMN) stimulated by lipopolysaccharide (LPS) produce platelet-activating factor (PAF) in a pattern characterized by an early and a delayed peak of synthesis. The early peak of PAF synthesis was due to a direct stimulation of these cells through mCD14 receptor as it was inhibited by anti-CD14 monoclonal antibody. The delayed and sustained peak of PAF synthesis was dependent on protein synthesis and cytokine production as shown by the inhibitory effect of cycloheximide on both MO and PMN, and of anti-tumour necrosis factor-alpha (anti-TNF-alpha) and of anti-interleukin-8 (anti-IL-8) neutralizing antibodies on MO and PMN respectively. IL-10 completely prevented this second, cytokine-dependent peak of PAF synthesis. In contrast, IL-10 markedly enhanced the first peak of PAF synthesis both in MO and PMN. Moreover, IL-10 was shown to modulate the production of superoxide anions (O2-) on both MO and PMN. As suggested by previous studies, IL-10 inhibited the delayed production of O2-. In the present study, we observed that IL-10 directly stimulated an early production of O2-. In addition, IL-10 enhanced the synthesis of O2- by MO and PMN challenged with LPS. The IL-10-induced O2- production was dependent, at least in part, from its effect on PAF synthesis, as it was inhibited by the PAF receptor antagonist WEB 2170. These results suggest that IL-10 may upregulate the early synthesis of PAF and O2- triggered by direct LPS stimulation, whereas it may downregulate the delayed production of these mediators.

Cell Culture Techniques↗
PubMed1997

Distinct pathogenic effects of group B coxsackieviruses on human glomerular and tubular kidney cells.

The six group B coxsackieviruses (CVBs) are highly prevalent human pathogens that cause viremia followed by involvement of different organs. Clinical and experimental evidence suggests that CVBs can induce kidney injury, but the susceptibility of human renal cells to these viruses is unknown. By using pure cultures of human glomerular and tubular cells, we demonstrated that all CVBs are capable of productively infecting renal cells of three different histotypes. Distinct pathogenic effects were observed. Proximal tubular epithelial cells and, to a lesser extent, glomerular podocytes were highly susceptible to CVBs; in both cases, infection led to cytolysis. In contrast, glomerular mesangial cells supported the replication of the six CVBs but failed to develop overt cytopathologic changes. Mesangial cells continued to produce infectious progeny for numerous serial subcultures (i.e., more than 50 days), especially with type 1, 3, 4, and 5 viruses. In the above cells, persistent infection induced the de novo synthesis of platelet-derived growth factor A/B and enhanced the release of transforming growth factor beta1/2. These two factors are important mediators of progression from glomerular inflammation to glomerulosclerosis. CVB replication appeared also to impair the phagocytic and contractile activity of mesangial cells. Loss of these properties--which are important in glomerular physiopathology--may contribute to the development of progressive nephropathy. The results show that CVBs induce distinct effects in different types of cultured renal cells and suggest that CVB infections may be associated with both acute and progressive renal injury.

Cytokines↗
PubMed1997

Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha.

We evaluated the role of an endogenous production of nitric oxide (NO) in the in vitro migration of endothelial cells and in the in vivo angiogenic response elicited by platelet-activating factor (PAF), tumor necrosis factor-alpha (TNF), and basic fibroblast growth factor (bFGF). The NO synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), but not its enantiomer D-NAME, prevented chemotaxis of endothelial cells induced in vitro by PAF and by TNF. The motogenic activity of TNF was also inhibited by WEB 2170, a specific PAF-receptor antagonist. In contrast, chemotaxis induced by bFGF was not prevented by L-NAME or by WEB 2170. Angiogenesis was studied in vivo in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model, the angiogenesis induced by PAF and TNF was inhibited by WEB 2170 and L-NAME but not by D-NAME. In contrast, angiogenesis induced by bFGF was not affected by L-NAME or by WEB 2170. TNF, but not bFGF, induced PAF synthesis within Matrigel. These results suggest that NO mediates the angiogenesis induced by PAF as well as that induced by TNF, which is dependent on the production of PAF. In contrast, the angiogenic effect of bFGF appears to be both PAF and NO independent.

Animals↗
PubMed1996

Determination of 1-O-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, platelet-activating factor and related phospholipids in biological samples by high-performance liquid chromatography--tandem mass spectrometry.

Combining normal-phase HPLC separation and tandem mass spectrometric detection, using an ion-spray HPLC-MS interface, a quantitative method for acyl-platelet activating factor (acyl-PAF), platelet-activating factor (PAF) and related phospholipids was developed. Mass spectra, positive ions, showed intense [M+H]+ ions; collision-induced dissociation of protonated molecular ions gave characteristic daughter ions corresponding to the polar head. Detection limits of 0.1-0.3 ng injected were obtained by multiple reaction monitoring. Samples of human endothelial cells treated with compounds modulating the levels of acyl-PAF and PAF have been analyzed by the present technique, proving that this approach is suitable for biochemical studies.

Cells, Cultured↗
PubMed1996

Plasmin promotes an endothelium-dependent adhesion of neutrophils. Involvement of platelet activating factor and P-selectin.

BACKGROUND: The adhesion of leukocytes to the endothelium and the edema of vessel wall may cause vascular reocclusion after thrombolytic therapy. The aim of this study was to evaluate the role of platelet activating factor (PAF) and P-selectin on the adherence of polymorphonuclear neutrophils (PMN) to the endothelium and of PAF on the increased vascular permeability induced by tissue-type plasminogen activator, streptokinase, and plasmin. METHODS AND RESULTS: We studied (1) the adhesion of 111Inlabeled PMN to human umbilical cord vein-derived cultured endothelial cells (HUVEC), (2) the transfer of 125I-labeled albumin across HUVEC monolayers, and (3) the adhesion of PMN to isolated bovine coronary arteries under flow conditions. It was found that the adhesion of PMN, induced by tissue-type plasminogen activator, streptokinase, and plasmin, correlated with the synthesis of PAF by HUVEC and was inhibited by WEB 2170, a PAF receptor antagonist. The adhesion of PMN was also inhibited by the treatment of HUVEC with anti-P-selectin antibodies or of PMN with soluble P-selectin or with anti-CD18 monoclonal antibodies. Plasmin also increased the permeability of HUVEC monolayers, an effect that was partially prevented by WEB 2170. Moreover, plasmin promoted the synthesis of PAF from isolated bovine coronary arteries and the adherence of PMN to the endothelium under flow conditions. The pretreatment of PMN with WEB 2170 or with soluble P-selectin prevented adhesion. CONCLUSIONS: The synthesis of PAF by endothelial cells at the site of plasmin generation and the endothelial expression of P-selectin may render the endothelial cell surface proadhesive for neutrophils and may favor a local increase in vascular permeability.

Animals↗
PubMed1996

Effects of recombinant human megakaryocyte growth and development factor on platelet activation.

The c-Mpl receptor for thrombopoietin and its recombinant related protein, the megakaryocyte growth and development factor (MGDF), is also expressed on circulating platelets. In the present study we evaluated the effect of MGDF on platelet aggregation in platelet-rich plasma (PRP) and in whole blood. The results obtained indicate that MGDF by itself did not affect platelet aggregation. However, when added before other agonists such as adenosine diphosphates (ADP), epinephrine (EPI), and thrombin (THR), it rendered platelets more sensitive. This "priming" effect of MGDF was dependent on the dose and on the time of platelet preincubation, and it occurred both in PRP and in whole-blood platelet aggregation. MGDF also "primed" the release of adenosine triphosphates and the production of thromboxane B2 by platelets stimulated with ADP, EPI, and THR. When added 15 seconds after the preincubation of platelets with subthreshold concentrations of ADP, EPI, and THR, MGDF exhibited a synergism with these agonists. Moreover, we observed a "priming" effect of MGDF on the phosphorylation of p-42 mitogen-activated protein kinase promoted by ADP, EPI, and THR. These observations suggest that thrombopoietin may play a physiologic role in modulating the response of platelets to several stimuli and thereby their hemostatic potential.

Adult↗
PubMed1996

Measurement of platelet-activating factor acetylhydrolase activity by quantitative high-performance liquid chromatography determination of coumarin-derivatized 1-O-alkyl-2-sn-lysoglyceryl-3-phosphorylcholine.

A sensitive method for determining platelet-activating factor acetylhydrolase (PAF-AH) activity in human serum, using high-performance liquid chromatography (HPLC) with a fluorimetric detection, is described. The method is based on the derivatization with 7-diethylaminocoumarin-3-carbonylazide of the 2-lyso-PAF, by-product of PAF-AH activity, extracted from the reaction mixture by phase partition into organic solvents. After 3 h of derivatization, the fluorescent derivatives were analyzed by HPLC on a reversed-phase column. The mobile phase was made up with a gradient between head solvent, composed of methanol:water (80:20, v/v) containing 0.25 g/liter choline chloride, and chloroform. Fluorescence detection was at excitation wavelength of 400 nm and at emission wavelength of 480 nm. The described chromatographic procedure is able to resolve and simultaneously quantitate the fluorescent derivatives of the C:18 and C:16 2-lysoPAF. The comparison with the classical radiometric determination of PAF-AH activity demonstrates that the herein described procedure is suitable for study of enzyme kinetics and changes occurring in physiological conditions such as pregnancy.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Role of tumor necrosis factor-alpha and platelet-activating factor in neoangiogenesis induced by synovial fluids of patients with rheumatoid arthritis.

The aim of the present study was to investigate in vivo in a mouse model the stimulation of neoangiogenesis by synovial fluids of patients with rheumatoid arthritis (RA) and to determine the role of tumor necrosis factor (TNF)-alpha and platelet-activating factor (PAF) in the formation of new vessels. Angiogenesis was studied in a mouse model in which Matrigel, injected subcutaneously, was used as a vehicle for the delivery of potential angiogenic stimuli. Synovial fluids of patients with RA but not with osteoarthritis (OA) were shown to induce neoangiogenesis. Since synovial fluid of patients with RA contained significantly higher levels of TNF-alpha-like bioactivity and of PAF than that of patients with OA, the role of these mediators was evaluated by using an anti-TNF-alpha neutralizing monoclonal antibody (mAb) and a PAF receptor antagonist, WEB 2170. When added to Matrigel, anti-TNF-alpha mAb and particularly WEB 2170 significantly reduced neoangiogenesis induced by synovial fluids of RA patients. Moreover, PAF extracted and purified from synovial fluid induced angiogenesis. These results suggest that the neoangiogenesis observed in rheumatoid synovitis may be due, at least in part, to the angiogenic effect of locally produced TNF-alpha and PAF.

Animals↗
PubMed1996

The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells.

The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.

Animals↗
PubMed1996

[Mechanisms of immune deposit formation in glomerulonephritis].

Recent experimental studies allowed the identification of several mechanisms of immune deposit formation, which are able to reproduce the morphological and clinical pattern of human glomerulonephritis. Moreover, it was shown that most of the lesions considered, in the past, as due to circulating immune complexes (IC), are instead caused by the "in situ" formation of IC. As a result of these studies, the following schematic classification was proposed: 1) immune deposits formed by glomerular localization of IC primarily formed in the circulation; 2) immune deposits formed "in situ" by reaction of circulating antibodies with fixed structural antigens; 3) immune deposits formed "in situ" by antibodies reactive with movable structural antigens; 4) immune deposits formed "in situ" by antibodies reactive with sequestered antigens leaking out of tissues; 5) IC formed "in situ" by antibodies reactive with exogenous or non-glomerular endogenous antigens planted in the glomeruli; 6) ANCA-associated glomerular disease.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1995

Lipopolysaccharide binding protein and CD14 modulate the synthesis of platelet-activating factor by human monocytes and mesangial and endothelial cells stimulated with lipopolysaccharide.

The biosynthesis of platelet-activating factor (PAF) during Gram-negative involves the interaction of LPS with the cells of the host. We have investigated the molecular mechanism that controls cell recognition and PAF biosynthetic response to LPS in human monocytes (MO), glomerular mesangial cells (MC), and HUVEC in culture. The synthesis of PAF by MO and MC involves two proteins, plasma LPS binding protein (LBP) and cell membrane CD14 (mCD14). As MO, MC were shown to express the mCD14 molecule by several mAbs. MO and mCD14-positive MC were stimulated to synthesize PAF either by the 63D3 and IOM-2 mAbs or by the natural ligand LBP-LPS complex. Moreover, LeuM3, 28C5, and 18E12 mAbs that were themselves unable to stimulate the synthesis of PAF blocked PAF synthesis initiated by LBP-LPS complex. LBP was required for synthesis of PAF by MO. In MC, which synthesize PAF also after stimulation by LPS alone, the LBP was shown to speed and significantly enhance the synthesis of PAF. The soluble form of CD14 (sCD14), when added to MO stimulated with LBP-LPS complexes, inhibited the synthesis of PAF possibly by competing with mCD14. In contrast, sCD14 was shown to be required for LPS-induced synthesis of PAF by HUVEC, which did not express mCD14. Therefore, membrane receptors (mCD14) and plasma soluble proteins (LBP and sCD14) may enable different human cell types to synthesize PAF after LPS stimulation.

Acute-Phase Proteins↗